Hello,
I have used an optional wash step in my miniprep said to remove endonucleases from DNA grown in strains of E. coli which produce endonucleases. The wash buffer was pre-warmed to 50C, which was optional. My plasmid yields were quite low when I used this buffer. Is it possible that the optional endonuclease-removing wash step lowers plasmid yields?
Thank you!
No optional washing will not affect the yeild..
Try to use more cells for plasmid isolation. Refer this link below
https://blog.addgene.org/optimizing-plasmids-101-plasmid-yields
I do not think the PB wash will lower the plasmid yield.
what is your yield?
No optional washing will not affect the yeild..
Try to use more cells for plasmid isolation. Refer this link below
https://blog.addgene.org/optimizing-plasmids-101-plasmid-yields
No it will not
To increase plasmid yield
1. Try heating your elutant i.e. molecular grade water to 37C to 50C before adding to column
2. Leave sitting on column for 5 min before spinning through
3. Now collect eluate add back to column wait 5 min and spin again
4. Also do not use 30ul as suggested. Go for upper limit of 50ul if miniprep
Finally as alluded to think about your starting culture and lag versus log versus stationary phase. You obviously want your cells ideally to be at top of exponential curve before harvesting cells. That means you should not have a protracted lag phase when plasmid can get spat out of host cells or be into stationary phase where cells start to break up. To do this
1. Pick your colony and grow during day in 5mls of LB without antibiotic selection
2. Take 10ul of that starter culture and add to 10ml over night culture of LB
3. Grow at 37C For first hour WITHOUT antibiotic
4. Now add antibiotic and grow for rest of night but no more than 14 hours
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